The selective advantage of mismatch repair loss in colonic stem cells

项目摘要 Lynch综合征（LS）是一种遗传性疾病，患者易患结直肠、子宫内膜、卵巢和子宫内膜癌。 其他癌症。LS是由DNA错配修复（MMR）基因中的遗传突变引起的， 也是15-30%散发性结直肠癌的基础。MMR功能丧失与1,000倍的 突变率的增加可能会增加重要癌基因和肿瘤抑制基因突变的风险。 MMR途径还激活细胞周期检查点和细胞死亡以响应外源DNA损伤， 然而，这种损伤反应在预防肿瘤发生中的作用尚不清楚。我们假设 失去这种MMR依赖性损伤反应的结肠干细胞（CSC）将获得选择性优势， 邻近的MMR-熟练细胞，特别是在诱变环境中，例如可以在结肠中发现。 我们预测，在DNA损伤增加的情况下，MMR的丧失将提高生存率，这有利于这些研究。 干细胞在竞争干细胞龛占有率。最终，这将导致生产更多的 高变肠细胞，增加癌症表型的转移率。检验这一假设， 由于缺乏合适的模型系统，以前存在问题。然而，人类的最新发展 结肠类器官和肠类模型现在允许我们通过以下方式研究MMR缺失对CSC动力学的影响： 以下目的：1）确定人胚胎干细胞（hESC）中MMR功能的丧失是否导致 在不存在或存在外源性DNA损伤剂的情况下的直接优势。我们将使用 CRISPR/Cas9介导的基因编辑敲除hESC中的MMR基因并检查其生长和 损害反应，并确定这些反应的机制。2)确定是否 MMR缺陷型CSC在结肠类器官和结肠类肠中具有选择性优势。为此目的， 我们将区分MMR熟练和缺陷的hESC为结肠类器官。作为一种补充模式，我们 还将从成人结肠组织样品中产生MMR敲除类肠蛋白。使用这两个系统，我们将 比较对外源性DNA损伤剂或致癌应激的反应。我们还将创建混合 含有MMR-熟练的和缺陷的细胞的类器官，并测试MMR-缺陷的细胞是否具有生长， 或在存在或不存在DNA损伤的情况下随着时间的推移而具有生存优势。3)确定MMR是否丢失 导致对体内CSC的选择性优势。我们将使用一种诱导型干细胞特异性基因敲除小鼠 Msh 2模型以产生镶嵌肠隐窝并测试MMR缺陷型CSC是否胜过野生型 CSC。总之，这些目标利用新的方法来研究MMR功能丧失的机制。 有助于肿瘤发生，提供信息，可能有助于解释疾病的发病率，同时指导 LS相关癌症的诊断、预防和治疗。

Project Summary Lynch syndrome (LS) is a hereditary disease that predisposes patients to colorectal, endometrial, ovarian and other cancers. LS is caused by inherited mutations in the DNA mismatch repair (MMR) genes, defects in which also underlie 15-30% of sporadic colorectal cancers. Loss of MMR function is associated with a 1,000-fold increase in mutation rate likely increasing the risk of mutation to important oncogenes and tumor suppressors. The MMR pathway also activates cell cycle checkpoints and cell death in response to exogenous DNA damage, however, the role of this damage response in preventing tumorigenesis is not known. We hypothesize that colonic stem cells (CSCs) that lose this MMR-dependent damage response will gain a selective advantage over neighboring MMR-proficient cells, particularly in a mutagenic environment such as may be found in the colon. We predict that loss of MMR will enhance survival under conditions of increased DNA damage, favoring these cells in a competition for stem cell niche occupancy. Ultimately, this will lead to the production of more hypermutable intestinal cells, increasing the penetrance of the cancer phenotype. Testing this hypothesis has been problematic previously due to lack of a suitable model system. However, the recent development of human colonic organoid and enteroid models now allow us to study the effects of MMR loss on CSC dynamics via the following aims: 1) Determine whether loss of MMR function in human embryonic stem cells (hESCs) leads to an immediate advantage in the absence or presence of exogenous DNA damaging agents. We will use CRISPR/Cas9-mediated gene editing to knock out the MMR genes in hESCs and examine their growth and damage response as well as determine the mechanism underlying those responses. 2) Determine whether MMR-deficient CSCs have a selective advantage in colonic organoids and colonic enteroids. For this purpose, we will differentiate MMR-proficient and deficient hESCs into colonic organoids. As a complementary model, we will also create MMR knock out enteroids from human adult colon tissue samples. Using both systems, we will compare the response to exogenous DNA damaging agents or oncogenic stress. We will also create mixed organoids containing MMR-proficient and deficient cells and test whether the MMR-deficient cells have a growth or survival advantage over time in the presence or absence of DNA damage. 3) Determine whether MMR loss leads to a selective advantage for CSCs in vivo. We will use an inducible, stem cell specific knockout mouse model of Msh2 to create mosaic intestinal crypts and test whether MMR-deficient CSCs outcompete wild-type CSCs. Together, these aims utilize novel approaches to study the mechanism by which loss of MMR function contributes to tumorigenesis providing information that may help explain disease penetrance while guiding the diagnosis, prevention and treatment of LS-associated cancers.